1. Field of the Invention
This invention relates to methods for detecting specific extracellular nucleic acid in plasma or serum fractions of human or animal blood associated with neoplastic or proliferative disease. Specifically, the invention relates to detection of nucleic acid derived from human viruses associated with human neoplasia, and to methods of detecting and monitoring extracellular viral nucleic acids found in the plasma or serum fraction of blood by using nucleic acid amplification with or without enrichment for viral DNA. In particular, the invention relates to the detection, identification, or monitoring of the existence, progression or clinical status of human neoplastic disease caused by or associated with viral infection through detection of viral nucleic acid in plasma or serum fractions. The invention permits the detection of extracellular, viral nucleic acid in the serum or plasma of humans or other animals recognized as having a neoplastic or proliferative disease or in individuals without any prior history or diagnosis of neoplastic or proliferative disease. The invention specifically provides methods for early identification of cervical carcinoma, cervical carcinoma in situ, cervical dysplasia, cervical intraepithelial neoplasia (CIN) and penile squamous cell carcinoma associated with infection by oncogenic human papillomavirus subtypes.
2. Background of the Related Art
Cervical carcinoma is a common form of malignancy afflicting women, arising from the squamous epithelium of the cervix. Much is known of the natural history of this disease. The vast majority of cases are attributable, at least in part, to an infection by a papillomavirus of the cervical epithelium. In particular, certain subtypes of human papillomavirus (HPV), including HPV subtypes 16, 18, 31, 33, and 35, are associated with cervical malignancy, where HPV infection seems to alter the epithelium to predispose an individual to the development of cancer. This alteration of the epithelium by viral infection initially leads to cervical premalignant states, specifically cervical dysplasia or cervical intraepithelial neoplasia (CIN). Cervical dysplasia/CIN is important for recognizing, diagnosing, and treating women at risk for developing cervical cancer, because surgical removal of dysplastic epithelium reduces and may even eliminate the risk of development of cervical cancer.
A prominent advance over the past several decades has been the reduction of the incidence of cervical carcinoma in the United States. In general, this advance has been attributed to the routine gynecologic examination and the introduction of the Pap smear, based upon cervical cytology. A scraping of the cervical epithelium is taken and eventually smeared on a glass slide for staining and microscopic examination. These procedures, typically carried out in a medical clinic by skilled personnel and forwarded to a laboratory for examination by technologists or specially developed machines, are useful in identifying early premalignant states. Surgical removal or destruction of the affected portion of the cervix can then be undertaken by a gynecologist, resulting in a marked reduction in the risk of development of carcinoma (although potentially associated with difficulty in child-bearing or infertility). Unfortunately, the Pap smear does not detect all instances of cervical dysplasia or premalignancy. The current acceptable rate of false negatives (i.e., women who do have dysplasia according to an expert panel of pathologists looking at tissue biopsies rather than smear samples, but are not diagnosed that way during the routine smear screening) is roughly 5–10% but recent studies suggest that the actual rate may be much higher. Furthermore, in approximately 7–8% of cases, the Pap smear demonstrates a typical squamous cells of undetermined significance (ASCUS). In an additional 20–30% of cases, the Pap smear may be insufficient for interpretation due to the presence of inflammatory cells.
Human papilloma virus (HPV) is a papillomavirus that very frequently infects a number of human tissues, including cervical tissue, and infection of cervical tissues is associated with the development of cervical carcinoma. The changes in cervical tissues attributable to viral infection are not visible to the unaided eye, but viral infection is evident at the microscopic level by changes in the epithelial cell's nucleus and cytoplasm that can be detected in a Pap smear. However, HPV infection cannot be detected in all cases of cervical dysplasia/pre-malignancy or even of cervical carcinoma. At least some of the failure to detect premalignancy using Pap smears as discusses above are the result of failure to detect HPV infection in cervical tissue samples.
HPV has many subtypes, and the subtypes may be distinguished by several methods. One commonly used method for evaluating exposure in humans to specific HPV subtypes, including those associated with cervical cancer, is by testing serum for antibodies against the viral subtype. Unfortunately, this method identifies both individuals with prior HPV exposure but without persistent infection and those with persistent infection, but does not distinguish between ongoing, persistent, and chronic HPV infections. This distinction is important, because persistent infection with HPV is associated with the greatest risk for development of cervical neoplasia. Because only a small percentage of women will develop cervical cancer within ten years of infectious exposure, active HPV infection is eliminated prior to the development of cervical premalignancy for most exposed women (Chua et al., 1996, Int. J. Cancer 68: 54–59). Serologic testing for HPV antibodies thus does not correlate well with the presence of cervical dysplasia, because it does not permit discrimination between previous HPV exposure and persistent HPV infection (Olsen et al., 1996, Int. J. Cancer 68: 415–419).
A second common method for screening women for specific HPV subtypes detects double stranded DNA viral genome (see, for example, Manos, U.S. Pat. No. 5,705,627; Bauer, U.S. Pat. No. 5,639,871; Bauer, U.S. Pat. No. 5,527,898; Manos, U.S. Pat. No. 5,283,171; Manos, U.S. Pat. No. 5,182,377; Gravitt et al., 1998, J. Clin. Microbiol. 36: 3020–3027). Those subtypes particularly associated with cervical cancer may be specifically amplified and detected using nucleic acid amplification methods, thereby identifying humans at high risk for the development of cervical carcinoma. Current methods for detecting HPV DNA entail evaluating cervical smear specimens directly for the viral DNA (see Silverstein, U.S. Pat. No. 5,814,448). This method is disadvantageous because the examiner may miss an infected region of the cervix, or the infected lesion may not be superficial and readily accessible, so that the test provides a false-negative result. Furthermore, as this method of testing for HPV DNA requires a gynecologic exam, DNA testing of cervical smears remains an imperfect method of screening the general female population because such exams are avoided by many women despite medical recommendations to the contrary.
In addition, HPV infection is a sexually transmitted disease, and males may carry the virus as asymptomatic carriers. Typically, cervical infection takes place at the time of sexual intercourse, and most of the risk factors (unprotected intercourse, multiple sexual partners, immunodeficiency, etc.) that apply to other sexually transmitted diseases also apply to HPV infection. Men may be infected by one or more HPV subtypes and can transmit one or more of these to their sexual partners. Although in most instances men serve only as carriers of the viral infection, in relatively rare cases they may develop squamous cell carcinoma of the penis in a manner analogous to cervical dysplasia and carcinoma in women. Both sexes may also develop anogenital warts, called condyloma accuminata, if infected by certain species of HPV, and these lesions may also be premalignant. Direct tissue swabs of the male urethra are not an effective general screening approach for detecting such carriers in the male population.
An attractive alternative approach to detecting HPV DNA in both women and men is testing peripheral blood for HPV DNA. However, it is not known in the art that papillomavirus DNA can be detected in the blood of individuals without cancer. Further, it is not known to the art whether papilloma virus can be detected in plasma and serum as an indication of persistent infection or risk for dysplasia or carcinoma.
Although many viruses have been shown to circulate in plasma and serum, the presence of systemically circulating extracellular HPV DNA has not been described in either the acute or chronic HPV infection. HPV infections tend to be localized as intracellular epithelial infections, and do not typically present with systemic clinical manifestations. Although metastatic circulating cervical cancer cells and leukocytes have been shown to have intracellularly incorporated HPV in cases of advanced cancer (Tseng et al., 1999, J. Clin. Oncol. 17: 1391–1396), detection of extracellular or free HPV DNA in blood is unknown in the art. To the contrary, the prior art suggests that some viruses are not readily detectable in the blood of infected individuals. An example is herpes simplex virus, which like HPV is a sexually-transmitted virus spread by mucosal contact. It was known in the art that, although herpes simplex virus could be found in blood serum of neonates with neonatal herpes simplex viremia, such could not be found in serum from older children having herpes simplex encephalitis, despite the presence of the virus in the cerebrospinal fluid of these older children (Kimura et al., 1991, J. Infect. Dis. 164: 289–293). In addition, HPV does not present with systemic viremia, and even for viruses in which viremia has been reported, the likelihood of detecting systemic viremia is greatest near the time of initial infection. This makes screening of women for the HPV virus doubly impractical, since any such screening would most likely be performed well beyond the period of initial exposure to the virus in the majority of cases.
One of the present inventors has shown that tumor-associated extracellular nucleic acids circulate in the plasma and serum of humans and animals (U.S. Ser. No. 08/818,058, the entire disclosure of which has been incorporated by reference). In particular, this application disclosed that tumor-associated DNA could be detected in the plasma and serum of humans and animals without cancer, enabling premalignancy to be detected, diagnosed, and monitored, and further, that individuals without cancer could be stratified or selected for further evaluation on the basis of the presence of mutant oncogene-derived DNA in blood plasma or serum.
Thus, there is a need in the art to determine whether HPV-specific nucleic acid can be detected in blood plasma or serum, and whether detection of HPV nucleic acid can be associated with the existence of cervical dysplasia or premalignancy. Improved screening methods having advantages over gynecological examinations and Pap smears would be enabled upon such a demonstration.